DESCRIPTION: One of the hallmarks of HIV is its extreme mutability, which leads to broad diversification of the viral quasispecies following infection. During mucosal transmission, the virus experiences the opposite phenomenon as well, undergoing a stringent narrowing of genotypic diversity, such that the majority of new infections are established by a single transmitted, founder (T/F) variant from the quasispecies. The physical determinants of transmission remain poorly defined. While many studies have traced T/F signature traits to Env, the surface glycoprotein that mediates infection, most have examined the biological consequences of underlying differences without being able to address what specifically differentiates one Env from another because classical methods are poorly suited to analyze structural variation of glycoproteins. The requirements for crystallography make it necessary to truncate and deglycosylate the glycoprotein, removing the very features such as variable loops and glycosylation that make each variant distinct and that can modulate structure within core, conserved regions. We propose to apply novel biophysical and structural approaches to compare the structure of Env glycoproteins from T/F variants of HIV with Env from donor variants using transmission pairs identified in a well-characterized mother-to-child-transmission (MTCT) study. We have developed the use of hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to probe intact glycoprotein structure under native conditions. This approach provides the sensitivity and resolution necessary to identify variant-specific differences, producing a fingerprint of local structural order and stability throughout the glycoprotein. In AIM 1, we will apply HDX-MS to analyze Env from matched transmission pairs identified in a Nairobi Breastfeeding MTCT study. The transmission pairs provide a powerful means to pinpoint sequence differences that may be linked with structural and phenotypic differences relevant to transmission of T/F variants. We hypothesize that structural properties in T/F Env may exist including greater stability that confer a selective advantage to T/F variants. We anticipate that greater Env stability may result in complexes that are resistant to inactivation In AIM 2, using infectivity assays, we will test whether T/F variants exhibit enhanced resistance to heat inactivation and inactivation by low pH and lactic acid, factors that may be present at sites of transmission such as the infant gastrointenstinal tract. The combination of structural biology and virological expertise in the Lee and Overbaugh labs applied to examine the matched transmission pairs can provide significant new insights into the physical determinants of virus transmissibility and provide structural details relevant to vaccine immunogen design.